A single proteinaceous antigen may contain a number of antigenic determinants that each include a number of specific epitopes that immunoreact with and are bound by antibodies directed or raised to the antigen as immunogen. Thus, the genome of a cell such as a pathogen may encode several antigenic determinants and epitopes that are recognized by a mammalian immune system as being foreign and that induce production of antibodies.
Where the antigenic determinants are part of a pathogen, antibody binding to particular epitopes may be of clinical importance in that such binding may lead to protection of the host animal in which the antibodies were raised. On the other hand, antibody binding to other epitopes may be of lesser importance to protection from the pathogen, but may provide information as to the strain of the immunizing pathogen or the stage of the disease state induced by the pathogen. It can therefore be seen that it may be clinically important or of scientific interest to be able to distinguish antibodies that immunoreact with and bind to one epitope of a given antigen from antibodies that immunoreact with and bind to one or more other epitopes of the same proteinaceous antigen
Early work with globular proteins seemed to indicate that antigenic determinants of those proteins were generally conformation-dependent; i.e., the epitopes were determined by both the amino acid residue sequence of the region and by the region's three-dimensional structure. For example, work reported by Arnon et al., Proc. Natl. Acad. Sci. USA, 62, 163 (1968) indicated that the so-called "loop" of the non-pathogenic, endogenous protein egg white lysosyme was a conformation-dependent antigenic determinant.
More recently, however, work by R. A. Lerner and co-workers using polypeptide immunogens whose sequences correspond to antigenic determinants of pathogen-related proteins has indicated that protection of a host animal from a pathogen may be obtained by use of vaccines containing immunogenic linear polypeptides that may assume many conformations. [See for example, Bittle et al., Nature, 298, 30 (1982); and Gerin et al., Proc. Natl. Acad. Sci. USA, 80, 2365 (1983).]The work of Lerner and co-workers thus appears to imply that a strict requirement of conformation-dependence of polypeptide immunogens, and therefore the existence of only conformation-dependent antigenic determinants and epitopes, is an incorrect assumption.
The heptatitis B virus (HBV) is associated worldwide with both acute and chronic diseases in man. A recent report has directly implicated that virus with hepatocellular carcinoma [Moriarty et al., Science, 227, 429 (1985); and Beasley et al., Lancet, 1129, Nov. 21, 1981.]
Immunologic markers encoded by the genome of HBV include the surface antigen (HBsAg), the core antigen (HBcAg), the core-derived HBeAg, and the so-called HBxAg reported on by Moriarty et al., above. Since HBsAg-specific antibodies are protective against HBV infection, virus-free HBsAg-containing envelopes present in the plasma of chronic carriers have served as a source of HBV vaccines.
The HBsAg is composed of a major polypeptide denominated p25, and its glycosylated form denominated Gp28. [peterson et al., Proc. Natl. Acad. Sci. USA, 74 1540 (1977).]The complete 226 amino acid sequence of the P25 polypeptide of HBsAg has been deduced from partial amino acid sequence data and from the neucleotide sequence of the viral gene (S) that encodes this polypeptide. [Valenzuela, Nature, 280, 815 (1979); Pasek et al., Nature, 282, 575 (1979); and Charnay et al., Nucleic Acids Res., 7, 335 (1979).]
Historically, additional higher molecular weight polypeptides associated with HBsAg were considered aggregates of P25 and GP28. However, P25 begins at the third possible translational initiation site of a larger open reading frame (ORF), and is preceded in phase by 163 or 174 condons (subtype-dependent) designated as the pre-S region. Tiollais et al., Science, 213, 406 (1980).
An HBV-associated 33-36 kilodalton (kD) glycoprotein (GP33) has been reported by Stibbe and Gerlich, J. Virol, 46, 626 (1983), and it was suggested that the sequence of GP33 starts at the second translational initiation signal of the ORF, which is 55 codons upstream of the third translation signal. [Cattaneo et al., Nature (London), 305, 336 (1983).]lt has also been reported that GP33 consists of the P25 sequence and an amino-terminal 55 amino acid residues encoded in what is now called the pre-S(2) region [Stibbe and Gerlich, above; and Machida et al., Gastorenterol., 86, 910 (1984).]
In support of that report, Neurath et al. Science, 224, 392 (1984) reported synthesis of a peptide encompassing the 26 amino-terminal amino acid residues of the pre-S(2) region. They reported that anti-peptide antibodies reacted with GP33. Recently, the products encoded from the first translational initiation site have been identified as P39/GP42, consisting of the GP33 sequence and the amino-terminal 108-119 amino acid residues encoded by the genome in the pre-S(1) region [Heermann et al., J. Virol., 52, 396 (1984).]
The fact that the pre-S region is conserved in all the described HBV DNA sequences and through evolution [Laub et al., J. Virol., 48, 271 (1983)]suggests a functional role for this region. This interpretation is enhanced by the observation that both the Gp42 and Gp33 polypeptides appear to be preferentially expressed in viremic carriers as opposed to carriers with minimal or no infectious virions in the blood. [Stibbe and Gerlich; and Heermann et al., above.]This suggests a correlation between viral replication and synthesis of the higher molecular weight polypeptides of HBsAg.
Additionally, the receptor for polymerized human albumin, which has been suggested to mediate viral attachment, has been reported to be localized on GP33. [Machida et al., Gastroenterol., 85, 320 (1982).]The implications of these recent findings relative to HBV vaccine development and mechanisms of immune-mediated viral clearance are apparent, and have prompted an investigation of the immune response to the S- and pre-S(2)-encoded GP33 polypeptide of HBsAg that is discussed hereinafter.